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Figure 2 | BMC Biochemistry

Figure 2

From: R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

Figure 2

Expression and interaction with endogenous Gβ5/RGS7 proteins of wild-type and mutant R7BP in PC12 cells. A. Co-immunoprecipitation of endogenous Gβ5/RGS7 with transfected R7BP constructs in PC12 cells. Cells were transfected with pcDNA3 (vector), wild type (wt) HA-R7BP, or the non-palmitoylated HA-R7BP-SS mutant cDNAs. The detergent lysate was immunoprecipitated using anti-HA antibody. The whole cell lysate (upper) and the precipitated proteins (lower) were analyzed by immunoblotting using anti-HA, anti-RGS7, and anti-Gβ5 (ATDG) antibodies as shown. The relative mobility of the major immunoreactive bands (in kDa) and the immunoglobulin heavy chain (HC) are indicated on the right. B. Fractionation analysis of PC12 cells transfected with wild type R7BP or its non-palmitoylated mutant. PC12 cells transfected with pcDNA3 (vector), wild type (wt) HA-R7BP, or the non-palmitoylated HA-R7BP-SS mutant were lysed, and after a low-speed spin to remove unbroken cells and nuclei, were separated into post-nuclear soluble fraction (cytosol) and particulate fractions (crude membranes) as shown, by ultracentrifugation. Localization of transfected HA-R7BP, and endogenous Gβ5 and RGS7 were detected by immunoblotting in both soluble and particulate fractions using appropriate antibody as indicated in Methods. These experiments were performed twice with similar results, as shown here.

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